Coronal Sections Of Rat Brain Research Articles

Neurotoxic Effects of Stanozolol on Male Rats' Hippocampi: Does Stanozolol cause apoptosis?

Stanozolol is an anabolic-androgenic steroid which is commonly abused by athletes for improved energy, appearance, and physical size. It has been previously shown to cause changes in behaviour and has various physical effects. Studies have previously been conducted on its neurotoxic effect on the central nervous system (CNS), which are typically psychological in nature. This study was performed to investigate the apoptotic effect of stanozolol on different parts of the rat hippocampus. Sixteen male Wistar rats were divided randomly into two groups (experimental and control). The experimental group received subcutaneous injections of stanozolol (5mg/kg/day) for consecutive 28 days, whereas the control group received saline using the same dosing schedule and administration route. After routine procedures, coronal sections of rat brain were stained with Toluidine blue and TUNEL for pre-apoptotic and apoptotic cell detection, respectively. In order to compare groups, the mean number of TUNEL-positive and pre-apoptotic neurons per unit area were calculated and analysed. Histopathological examination revealed that the mean number of pre-apoptotic and apoptotic neurons in the CA1, CA2, CA3 and DG areas of the hippocampus were significantly increased in the stanozolol treated group. In conclusion, stanozolol abuse may induce pre-apoptotic and apoptotic cell formation in different regions of the hippocampus.

Short N-terminal galanin fragments are occurring naturally in vivo

The galanin family currently consists of four peptides, namely galanin, galanin-message associated peptide, galanin-like peptide and alarin. Unlike galanin that signals through three different G protein-coupled receptors; GAL1, GAL2, and GAL3, binding at its N-terminal end, the cognate receptors for other members of the galanin family are currently unknown. Research using short N-terminal galanin fragments generated either by enzymatic cleavage or solid-phase synthesis has revealed differences in their receptor binding properties exerting numerous biological effects distinct from galanin(1-29) itself. Our studies on tissue extracts derived from rat small intestine and bovine gut using chromatographic techniques and sensitive galanin(1-16)-specific radioimmunoassay revealed the presence of immunoreactive compounds reacting with antiserum against galanin(1-16) distributed in distinct elution volumes. These results suggested a possible presence of short N-terminal galanin fragments also in vivo. Moreover, employing immunoaffinity chromatography and reverse-phase high performance liquid chromatography (HPLC) followed by mass spectrometry allowed specific enrichment of these immunoreactive compounds from rat tissues and identification of their molecular structure. Indeed, our study revealed presence of several distinct short N-terminal galanin sequences in rat tissue. To prove their receptor binding, four of the identified sequences were synthetized, namely, galanin(1-13), galanin(1-16), galanin(1-20), galanin(6-20), and tested on coronal rat brain sections competing with 125I-labeled galanin(1-29). Our autoradiographs confirmed that galanin(1-13), galanin(1-16), and galanin(1-20) comprehensively displaced 125I-galanin(1-29) but galanin(6-20) did not. Here we show, for the first time, that short N-terminal galanin fragments occur naturally in rat tissues and that similar or identical galanin sequences can be present also in tissues of other species. Biological significanceThis study is first to provide an evidence of the presence of short N-terminal galanin fragments in vivo in a biological system and provides further foundations for the previous studies using synthetized short N-terminal galanin fragments.

A Novel Method for Determination of Drug Distribution in Rat Brain Tissue Sections by LC/MS/MS: Functional Tissue Microanalysis

Determination of drug distribution in brain and other tissues is important in pharmaceutical research. Tissue drug levels need to be determined routinely as they are usually diagnostic for both efficacy and toxicity. Determination of tissue levels in small organ subregions is frequently performed due to important functional considerations. These measurements have traditionally been very tedious requiring extensive dissection and specimen pooling to achieve detection of analytes of interest. Direct and indirect methods utilizing mass spectrometry have been reported for detection of analytes in tissue specimens. Typically, these require very specialized MS or sampling equipment and are only partially successful due to analyte response. We have developed a novel approach for quantitation of tissue sections called Functional Tissue Microanalysis (FTM) in which small circular samples are removed from subregions of interest, extracted and analyzed by conventional LC/MS/MS utilizing electrospray ionization. This allows direct measurement of regional concentrations without dissection and homogenization of tissue specimens as many subregions can be sampled from a single mounted section. Utilization of the FTM approach for analysis of both sagittal and coronal rat brain sections is shown for quantitation of raclopride and rimonabant. Reproducibility of this approach and comparison to conventional methods is reported.

A minimalist approach to MALDI imaging of glycerophospholipids and sphingolipids in rat brain sections

Matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) is a powerful tool that has allowed researchers to directly probe tissue molecular structure and drug content with minimal manipulations, while maintaining anatomical integrity. In the present work glycerophospholipids and sphingolipids images were acquired from 16-μm thick coronal rat brain sections using MALDI-MS. Images of phosphatidylinositol 38:4 (PI 38:4), sulfatide 24:1 (ST 24:1), and hydroxyl sulfatide 24:1 (ST 24:1 (OH)) were acquired in negative ion mode, while the images of phosphatidylcholine 34:1 (PC 34:1), potassiated phosphatidylcholines 32:0 (PC 32:0 + K +) and 36:1 (PC 36:1 + K +) were acquired in positive ion mode. The images of PI 38:4 and PC 36:1 + K + show the preferential distribution of these two lipids in gray matter; and the images of two sulfatides and PC 32:0 + K + show their preferential distribution in white matter. In addition, the gray cortical band and its adjacent anatomical structures were also identified by contrasting their lipid makeup. The resulting images were compared to lipid images acquired by secondary ion mass spectrometry (SIMS). The suitability of TLC sprayers, Collison Nebulizer, and artistic airbrush were also evaluated as means for matrix deposition.

MALDI‐ion mobility‐TOFMS imaging of lipids in rat brain tissue

While maintaining anatomical integrity, matrix assisted laser desorption/ionization mass spectrometry (MALDI-MS) has allowed researchers to directly probe tissue, map the distribution of analytes and elucidate molecular structure with minimal preparation. MALDI-ion mobility (IM)-orthogonal time-of-flight mass spectrometry (oTOFMS) provides an advantage by initially separating different classes of biomolecules such as lipids, peptides, and nucleotides by their IM drift times prior to mass analysis. In the present work the distribution of phosphatidlycholine and cerebroside species was mapped from 16 microm thick coronal rat brain sections using MALDI-IM-oTOFMS. Furthermore, the use of gold nanoparticles as a matrix enables detection of cerebrosides, which although highly concentrated in brain tissue, are not easily observed as positive ions because of intense signals from lipids such as phosphatidlycholines and sphingomyelins.

Comparison of [ 3H]-(2 S,4 R)-4-methylglutamate and [ 3H] d-aspartate as ligands for binding and autoradiographic analyses of glutamate transporters

While studies with [ 3H] d-aspartate ([ 3H] d-Asp) illustrate specific interactions with excitatory amino acid transporters (EAATs), new insights into the pharmacological characteristics and localization of specific EAAT subtypes depend upon the availability of novel ligands. One such ligand is [ 3H]-(2 S,4 R)-4-methylglutamate ([ 3H]4MG) which labels astrocytic EAATs in homogenate binding studies. This study examined the utility of [ 3H]4MG for binding and autoradiography in coronal sections of rat brain. Binding of [ 3H]4MG was optimal in 5 mM HEPES buffer containing 96 mM NaCl, pH 7.5. Specific binding of [ 3H]4MG exhibited two components, but was to a single site when glutamate receptor (GluR) sites were masked with kainate (KA; 1 μM): t 1/2 ∼ 5 min, K d 250 nM and B max 5.4 pmol/mg protein. Pharmacological studies revealed that [ 3H]4MG, unlike [ 3H] d-Asp, labeled both EAAT and ionotropic GluR sites. Further studies employed 6-cyano-7-nitroquinoxaline (30 μM) to block GluR sites, but selective EAAT ligands displayed lower potency than expected for binding to transporters relative to drugs possessing mixed transporter/receptor activities. Autoradiography in conjunction with densitometry with [ 3H]4MG and [ 3H] d-Asp revealed wide, but discrete distributions in forebrain; significant differences in binding levels were found in hippocampus, nucleus accumbens and cortical sub-areas. Although EAAT1 and EAAT2 components were detectable using 3-methylglutamate and serine- O-sulphate, respectively, the majority of [ 3H]4MG binding was to KA-related sites. Overall, in tissue sections [ 3H]4MG proved unsuitable for studying the autoradiographic localization of EAATs apparently due to its inability to selectively discriminate Na +-dependent binding to Glu transporters.

Effects of morphine withdrawal on μ-opioid receptor-stimulated guanylyl 5′-[γ-[ 35S]thio]-triphosphate autoradiography in rat brain

Abstinence from chronic morphine results in characteristic withdrawal symptoms in humans and experimental animals. Despite a large number of studies, the cellular mechanisms underlying opiate withdrawal symptoms are not clearly understood, in particular, the regulation of μ-opioid receptor function during this process. The present study investigated the μ-opioid receptor-stimulated G-protein activity using guanylyl 5′-[γ-[ 35S]thio]-triphosphate ([ 35S]-GTPγS) autoradiography. [ 35S]-GTPγS binding was performed using coronal rat brain sections (20 μm) in the presence or absence of the μ-opioid selective agonist [ d-Ala 2, N-MePhe 4Gly 5-ol] enkephalin (DAMGO). In experiment 1, rats (male, Sprague–Dawley) were injected every 12 h with increasing doses of morphine (5–100 mg/kg, s.c.) for 12 days; a separate group of rats which received saline injections served as control. Opiate withdrawal was induced by abstinence from morphine. Thirty-six hours after the last morphine injection, spontaneous withdrawal symptoms were assessed. Rats were then decapitated and brains rapidly removed. In experiment 2, withdrawal symptoms were precipitated with the opioid receptor antagonist naloxone (1 mg/kg). Brains were taken at 5, 10, 20 and 60 min after naloxone injection. In experiment 3, morphine dependence was induced by implantation of three morphine pellets (75 mg per pellet). After 7 days, withdrawal symptoms were precipitated by naloxone (1 mg/kg) and brains were removed 30 min after naloxone injection. [ 35S]-GTPγS binding was measured in the locus coeruleus, nucleus parabrachialis, nucleus accumbens and central nucleus of amygdala. Although clear withdrawal symptoms were observed in all morphine-withdrawn rats, no significant changes in [ 35S]-GTPγS binding were detected in animals undergoing withdrawal. The present lack of differences between morphine-withdrawn and control rats indicates that μ-opioid receptor-stimulated G-protein activity is not modulated by chronic morphine administration and is not involved in the expression of opiate withdrawal.

Evidence for the Differential Distribution of at Least Two Adrenomedullin Receptor Subtypes in the Rat Brain

We recently reported that specific [I]human Adrenomedullin13-52 (hADM13-52) binding sites are very discretely distributed in the rat brain[1]. Furthermore, in rat brain membrane homogenates, we suggested the existence of at least two populations of [I]hADM13-52 receptor binding sites[2]. In the present study, we investigated the differential distribution of specific [I]hADM1352 sites that are sensitive and insensitive to hADM22-52 in the rat brain. Adjacent coronal rat brain sections were incubated with 35 pM [I]hADM13-52 in the presence of increasing concentrations of hADM22-52. As previously reported, specific [I]hADM13-52 sites were observed in the choroid and linings of ventricles, basal amygdaloid nucleus, neuronal lobe of the pituitary gland, trigeminal nucleus, and cerebellum. Specific [I]hADM13-52 binding is fully inhibited by 1 μM hADM22-52 in all these brain regions except in the cerebellum. In fact, hADM22-52 was able to inhibit only 70% of specific [I]hADM13-52 binding sites in the rat cerebellum. These data suggest for the first time that the rat cerebellum contains at least two populations of ADM receptors, one that is sensitive to hADM22-52 while the other is resistant. This characterization of cerebellar [I]hADM13-52 / hADM22-52 -insensitive sites is currently under investigation. Supported by grant from the CIHR to RQ.

Autoradiographic localization of 125I[Tyr14]orphanin FQ/nociceptin and 125I[Tyr10]orphanin FQ/nociceptin(1–11) binding sites in rat brain

The endogenous ligand for the orphan opioid receptor, orphanin FQ/nociceptin (OFQ), has recently been characterized. The OFQ peptide sequence contains paired basic amino acids, suggesting the possibility of posttranslational processing to a peptide containing the first 11 amino acids of the OFQ peptide. This peptide has been reported in the brain and it has a unique pharmacology. In the present study, we compared the autoradiographic distribution of 125I[Tyr14]OFQ and 125I[Tyr10]OFQ(1–11) in coronal rat brain sections. Nonspecific binding was defined with unlabeled OFQ or OFQ(1–11), respectively. Both radioligands demonstrated high levels of specific binding (>95% of total binding), with no appreciable binding in white matter areas with either ligand. 125I[Tyr14]OFQ binding was widely distributed throughout the rat brain. In contrast, 125I[Tyr10]OFQ(1–11) binding was more restricted. The highest 125I[Tyr14]OFQ binding levels measured in this study were found in the locus coeruleus, an area which contained very low 125I[Tyr10]OFQ(1–11) binding. Both ligands labeled the cortex, hippocampus and amygdala. In the thalamus, 125I[Tyr14]OFQ binding was prominent in most nuclei, whereas 125I[Tyr10]OFQ(1–11) binding was restricted to the midline thalamus. 125I[Tyr14]OFQ binding was heavy in the suprachiasmatic hypothalamus, and moderate in other hypothalamic nuclei. 125I[Tyr10]OFQ(1–11) binding in the hypothalamus, however, was present mainly in the ventromedial hypothalamic nucleus. Lower binding levels of both ligands were found in the caudate putamen. The distinct autoradiographic patterns of these two ligands are consistent with different binding sites, which might help explain their different functional activities. J. Comp. Neurol. 423:319–329, 2000. © 2000 Wiley-Liss, Inc.

Autoradiographic localization of125I[Tyr14]orphanin FQ/nociceptin and125I[Tyr10]orphanin FQ/nociceptin(1-11) binding sites in rat brain

The endogenous ligand for the orphan opioid receptor, orphanin FQ/nociceptin (OFQ), has recently been characterized. The OFQ peptide sequence contains paired basic amino acids, suggesting the possibility of posttranslational processing to a peptide containing the first 11 amino acids of the OFQ peptide. This peptide has been reported in the brain and it has a unique pharmacology. In the present study, we compared the autoradiographic distribution of (125)I[Tyr(14)]OFQ and (125)I[Tyr(10)]OFQ(1-11) in coronal rat brain sections. Nonspecific binding was defined with unlabeled OFQ or OFQ(1-11), respectively. Both radioligands demonstrated high levels of specific binding (>95% of total binding), with no appreciable binding in white matter areas with either ligand. (125)I[Tyr(14)]OFQ binding was widely distributed throughout the rat brain. In contrast, (125)I[Tyr(10)]OFQ(1-11) binding was more restricted. The highest (125)I[Tyr(14)]OFQ binding levels measured in this study were found in the locus coeruleus, an area which contained very low (125)I[Tyr(10)]OFQ(1-11) binding. Both ligands labeled the cortex, hippocampus and amygdala. In the thalamus, (125)I[Tyr(14)]OFQ binding was prominent in most nuclei, whereas (125)I[Tyr(10)]OFQ(1-11) binding was restricted to the midline thalamus. (125)I[Tyr(14)]OFQ binding was heavy in the suprachiasmatic hypothalamus, and moderate in other hypothalamic nuclei. (125)I[Tyr(10)]OFQ(1-11) binding in the hypothalamus, however, was present mainly in the ventromedial hypothalamic nucleus. Lower binding levels of both ligands were found in the caudate putamen. The distinct autoradiographic patterns of these two ligands are consistent with different binding sites, which might help explain their different functional activities.

Structural organization of the perivascular astrocyte endfeet and their relationship with the endothelial glucose transporter: A confocal microscopy study

Despite the increasing evidence for a prominent role played by the perivascular endfeet of astrocytes in the functional metabolic coupling between astrocytes and neurons, a clear picture of their spatial organization is still lacking. To examine the three-dimensional structure of the astrocyte endfeet and their relationships with the endothelial cells, coronal rat brain sections immunolabeled for the two astroglial markers [glial fibrillary acidic protein (GFAP)/S-100β] and the endothelial glucose transporter (GLUT1) were analyzed under the confocal microscope. Double immunolabeling of GFAP and S-100β showed numerous well-defined astrocytes sending one or more endfeet to the vasculature. Examination of GFAP immunolabeling at higher magnification showed that these endfeet consist of well-defined rosette-like structures lying on the vessel wall. Double immunostaining of GFAP and GLUT1 showed that the endothelial cells were the main targets of these repeated geometrical units formed by the astrocyte endfeet. When three-dimensional images were reconstructed, obvious privileged anatomical relationships were observed between endfeet and individual endothelial cells. These anatomical data provide strong support for the involvement of astrocytes in cerebral metabolic coupling. The finger-like appearance of astrocyte endfeet could allow direct metabolic exchanges between intracerebral vessels and non-glial elements such as nerve terminals. GLIA 23:1–10, 1998. © 1998 Wiley-Liss, Inc.

Structural organization of the perivascular astrocyte endfeet and their relationship with the endothelial glucose transporter: A confocal microscopy study

Despite the increasing evidence for a prominent role played by the perivascular endfeet of astrocytes in the functional metabolic coupling between astrocytes and neurons, a clear picture of their spatial organization is still lacking. To examine the three-dimensional structure of the astrocyte endfeet and their relationships with the endothelial cells, coronal rat brain sections immunolabeled for the two astroglial markers [glial fibrillary acidic protein (GFAP)/S-100beta] and the endothelial glucose transporter (GLUT1) were analyzed under the confocal microscope. Double immunolabeling of GFAP and S-100beta showed numerous well-defined astrocytes sending one or more endfeet to the vasculature. Examination of GFAP immunolabeling at higher magnification showed that these endfeet consist of well-defined rosette-like structures lying on the vessel wall. Double immunostaining of GFAP and GLUT1 showed that the endothelial cells were the main targets of these repeated geometrical units formed by the astrocyte endfeet. When three-dimensional images were reconstructed, obvious privileged anatomical relationships were observed between endfeet and individual endothelial cells. These anatomical data provide strong support for the involvement of astrocytes in cerebral metabolic coupling. The finger-like appearance of astrocyte endfeet could allow direct metabolic exchanges between intracerebral vessels and non-glial elements such as nerve terminals.

Distribution of dopamine beta-hydroxylase-like immunoreactive fibers within the shell subregion of the nucleus accumbens.

The nucleus accumbens (Acb) can be divided into distinct subfields, delineated on the basis of histochemical markers as well as by afferent and efferent projection patterns. The shell subregion has reciprocal relationships with a variety of limbic areas and brainstem autonomic structures, and has been suggested to participate in motivation-related processes, including reward, stress, and arousal. The locus coeruleus (LC)-noradrenergic system has similarly been implicated in the modulation of behavioral state and stress-related processes, and previous studies have demonstrated reciprocal projections between the locus coeruleus and Acb shell. To better understand the anatomical substrate through which LC could influence activity within Acb shell, immunohistochemical methods were used to visualize the extent and the distribution of noradrenergic axons within this structure. Coronal sections of rat brain were processed to visualize immunoreactivity for the norepinephrine synthetic enzyme dopamine beta-hydroxylase (DBH), a specific marker for noradrenergic processes. In some cases, alternate sections were processed for immunohistochemical localization of substance P, in order to delineate core, shell, and pallidal compartments. Moderate-to-dense DBH-like immunoreactivity (DBHir) was found in approximately the caudal half of the shell subregion, particularly in caudalmost (septal pole) and ventral zones. The innervation of the septal pole was contiguous with a dense innervation of the bed nucleus of the stria terminalis. Few immunoreactive fibers were observed in the caudate-putamen, Acb core, or rostral Acb shell. Many DBHir fibers within the shell region were highly arborized with numerous varicosities, features indicative of terminal fields. These observations suggest noradrenergic systems might modulate certain processes associated with stress, behavioral state, or reinforcement via actions within the Acb shell.

Localization of α2A- and α2B-Adrenergic Receptor Subtypes in Brain

Recent studies have shown that all three subtypes of alpha2-adrenergic receptor (alpha2-AR) are found in brain. The purpose of this study was to map the subtype localization of the alpha2A- and alpha2B-ARs in brain structures. RNase protection shows that both the alpha2A- and alpha2B-ARs are detectable in cortex, cerebellum, pons-medulla, and hypothalamus. We tested probes derived from the alpha2A- and alpha2B-AR cDNAs on cell lines that express each of the alpha2-AR subtypes to establish the subtype specificity of these probes for in situ hybridization. Then we used the alpha2A- and alpha2B-AR probes for in situ hybridization on sagittal and coronal sections of rat brain. Both alpha2A and alpha2B mRNA were detected throughout the brain. Overall, there appears to be a greater expression of message for alpha2A- than alpha2B-AR in most brain areas, with the exception of the thalamus. Developing these probes for in situ hybridization is an important step for further studies on the exact role of the alpha2-AR subtypes in neurons that modulate cardiovascular function.

Localization of serotonin subtype 6 receptor messenger RNA in the rat brain by in situ hybridization histochemistry.

The serotonin receptor subtype 6, which raises intracellular cyclic AMP via stimulatory G-proteins, has recently been cloned and characterized. To determine the distribution of serotonin subtype 6 messenger RNA, in situ hybridization was performed in coronal sections of rat brain. 35S-labeled riboprobe, complementary to the 5' non-coding region of the serotonin subtype 6 messenger RNA, and a 33P-labeled riboprobe complementary to its 3' non-coding region, were used for hybridization. Serotonin subtype 6 receptor message was found in serotonin projection fields, rather than regions of serotonin-containing cell bodies, suggesting that the receptor is mainly postsynaptic. Hybridization signal was highest in olfactory tubercle, as well as prominent in the striatum, nucleus accumbens, dentate gyrus, and CA1, CA2 and CA3 of the hippocampus. Less intense hybridization was observed in cerebellum, some diencephalic nuclei, the amygdala, and layers 2, 3, 4 and 6 of the cortex. This pattern of hybridization was observed with both probes, but not when sense transcripts were used. Because the serotonin subtype 6 receptor has a high affinity for the atypical antipsychotic clozapine, and because striatum and nucleus accumbens are proposed sites of antipsychotic drug effects, the possibility is raised that this receptor may play an important role in mediating the effects of the atypical antipsychotic agents.

Quantitative autoradiography of the serotonin transporter to assess the distribution of serotonergic projections from the dorsal raphe nucleus.

The binding of 3H-CN-IMI to 5-HT uptake sites, as measured by quantitative autoradiography, was used as a marker of serotonergic neurons. Within the dorsal raphe nucleus the binding of 3H-CN-IMI was compared in adjacent coronal sections of rat brain to the binding of 3H-DPAT to 5-HT1A receptors, which have a known somatodendritic localization. The heterogeneous pattern of binding of these two radioligands within the dorsal raphe nucleus was similar and corresponded to the distribution of serotonergic cell bodies as visualized by 5-HT immunohistochemistry. Intracerebroventricular administration of 5,7-dihydroxytryptamine (5,7-DHT), which caused a dramatic loss of 5-HT immunoreactivity and 3H-DPAT binding to 5-HT1A receptors, resulted in a marked reduction of 3H-CN-IMI binding in this nucleus. Treatment of rats with a dose of para-chloroamphetamine (PCA) which has been reported to selectively lesion serotonergic processes arising from the dorsal raphe nucleus, while sparing serotonergic cell bodies and projections from the median raphe nucleus, did not alter the binding of 3H-DPAT or 3H-CN-IMI in the dorsal raphe nucleus; serotonergic cell bodies appeared morphologically unaffected. The lack of effect of PCA treatment on the binding of 3H-DPAT and 3H-CN-IMI is consistent with a somatodendritic localization of the 5-HT transporter in the dorsal raphe nucleus. PCA treatment appeared to produce a moderate loss of serotonergic innervation in serotonergic terminal field areas as visualized by serotonin immunohistochemistry. The reductions in 3H-CN-IMI binding observed in terminal field areas (24 to 69%) following treatment of rats with PCA did not reflect a marked differential innervation of forebrain areas by the dorsal and medial raphe nuclei as expected from previous biochemical studies, and were not entirely consistent with the findings of neuroanatomical studies using histochemical techniques. Site-specific injection of 5,7-DHT into the dorsal raphe nucleus produced an 80 +/- 11% reduction in the binding of 3H-CN-IMI in this nucleus, whereas the binding of 3H-CN-IMI in the median raphe nucleus was not reduced.(ABSTRACT TRUNCATED AT 400 WORDS)

Rapid Gelatin Embedding Procedure for Frozen Brain Tissue Sectioning

Abstract Traditional procedures used to embed formalin fixed brain tissue in gelatin before frozen sectioning require soaking periods as long as 72 hr. By washing formalin from tissue surface layers and avoiding excessive soaking times, which can prematurely harden gelatin, embedding time can be reduced to 90 min. This simple procedural modification allows the cutting of 30 μm coronal rat brain sections, for example, on a clinical microtome without shredding or other distortion that would interfere with microscopic examination. (The J Histotechnol 15:121, 1992)

Effects of propentofylline on adenosine A 1 and A 2 receptors and nitrobenzylthiojnosine-sensitive nucleoside transporters: Quantitative autoradiographic analysis

Previous studies have demonstrated that the xanthine compound, propentofylline, has beneficial effects in models of cerebral ischemia and can enhance some and exhibit other effects of adenosine. We investigated the in vitro effects of propentofylline and its hydroxy metabolite, A72 0287, on the binding of( 3H]cyclohexyladenosine ([ 3H]CHA), [ 3H]2-[p-(2-carbonyl-ethyl)-phenylethyl-amino]-5'-N-ethylcarboxamido adenosine ([ 3H]CGS 21680) and [ 3H]nitrobcnzylthioinosine ([ 3H]NBMPR) to adenosine A 1 and A 2 receptors and NBMPR-sensitive nucleoside transporters, respectively, in 10-μm coronal rat brain sections. Both xanthines had micromolar affinity for each of these sites with approximately 10-fold lower affinity for A 2 receptors than for A 1 receptors and [ 3H]NBMPR binding sites. Saturation analysis of [ 3]CHA or [ 3h]cgs 21680 binding in the presence of increasing concentrations of propentofylline produced significant increases in K d values without affecting B max values; thus propentofylline is a competitive inhibitor at a 1 and A 2 receptors. The effects on A 2 receptors apparently require higher concentrations (K i approximately 200 μM) than the effects on A 1 receptors (K i approximately 20 μM). Propentofylline was also found to be a competitive inhibitor of [ 3h]nbmpr binding. Therefore we conclude that propentofylline interacts with adcnosine-responsive systems to increase interstitial adenosine concentrations and to selectively inhibit A 1 receptors.

CENTRAL INSULIN ADMINISTRATION REDUCES NEUROPEPTIDE Y mRNA EXPRESSION IN THE ARCUATE NUCLEUS OF FOOD-DEPRIVED LEAN (Fa/Fa) BUT NOT OBESE (fa/fa) ZUCKER RATS

By acting in the brain, insulin suppresses food intake, whereas neuropeptide Y (NPY) has the opposite effect. Since fasting increases NPY gene expression in the hypothalamic arcuate nucleus (ARC) and also lowers circulating insulin levels, we hypothesized that the anorexiant effect of insulin could result from insulin inhibition of NPY gene transcription in the ARC. Therefore, we determined whether the administration of insulin (200 mU per 12 hrs) into the 3rd cerebral ventricle of lean (Fa/Fa) female Zucker rats (n = 5) during 48 hrs of food deprivation reduces the expression of preproNPY mRNA in the ARC compared to vehicle-treated controls (n = 5). Coronal sections of rat brain were hybridized with an oligonucleotide probe complementary to preproNPY mRNA and apposed to x-ray film. Hybridization was quantified in both the ARC and the hippocampal dentate gyrus by computerized image analysis of the resulting autoradiographs. Central insulin significantly reduced the area of hybridization in the ARC (0.235 +/- 0.017 mm2; mean +/- SE) compared to vehicle-treated controls (0.331 +/- 0.037 mm2; p less than 0.05), but was without effect in the hippocampus. Thus, insulin reduced the expression of mRNA for NPY specifically in the ARC. Since the genetically obese (fa/fa) Zucker rat is insensitive to the anorexiant effect of insulin and over-expresses NPY in the ARC, we next tested the hypothesis that insulin does not suppress NPY mRNA expression in the ARC of these rats. Consistent with this hypothesis, central insulin administration to obese Zucker rats during 48 hrs of food deprivation (n = 6) did not lower hybridization area in the ARC compared to vehicle alone (n = 4) (0.286 +/- 0.036 vs. 0.248 +/- 0.019 mm2; p greater than 0.05). We conclude that insulin suppresses the expression of mRNA for NPY in the ARC of fasted lean but not obese Zucker rats. Regulation of hypothalamic NPY gene expression by insulin may account for its anorexiant effect, and a defect in this action may contribute to certain forms of obesity.

The regional distribution of sulphonylurea binding sites in rat brain

Sulphonylureas such as glibenclamide, which are used in the treatment of Type-2 diabetes, are inhibitors of ATP-sensitive potassium channels. These channels link cellular metabolism to membrane electrical activity and it is likely that they are closely associated with glibenclamide binding sites. Quantitative autoradiography was used to localize high-affinity [ 3H]glibenclamide binding sites in coronal sections of rat brain. The relative density of binding sites was found to correlate well with the relative capacity of sites determined in homogenate assays. There was no evidence of any variation of affinity between brain regions. The highest levels of binding were found in the substantia nigra with high levels in the globus pallidus, cerebral cortex, hippocampus and caudate-putamen, intermediate levels in the cerebellum, and low levels in the hypothalamus and pons. The density of [ 3H]glibenclamide binding sites was low in glucose-responsive brain regions, known to contain ATP-sensitive potassium channels that are inhibited by sulphonylureas. However, higher densities were associated with brain regions (often limbic structures) active during temporal lobe epilepsy. In at least two of these structures, the CA3 region of the hippocampus and the substantia nigra, it is probable that these sites are coupled to ATP-sensitive potassium channels. These results are discussed with reference to the reported actions of ATP-sensitive potassium channels on CNS function.